Polyelectrolyte multilayers (PEMs) have many potential applications in tissue engineering and regenerative medicine. However, the softness of biocompatible PEMs results in limited cell adhesion. A novel strategy for the enhancement of cell adhesion on PEMs based on thermal annealing is presented here. The impact of thermal annealing at 37 ºC of poly-l-lysine (PLL) and alginate (Alg) polyelectrolyte multilayers on the adhesion of human lung cancer A549 and myoblast C2C12 cell lines is studied. The properties of the PEMs after annealing are characterized by means of the quartz crystal microbalance with dissipation, atomic force microscopy, atomic force spectroscopy, zeta potential, and contact angle measurements. After annealing, PLL/Alg PEMs become smoother displaying an increase in stiffness. Furthermore, PEMs become more hydrophobic, with an increase in contact angle from 36° to 90°. Additionally, the surface charge decreases and protein deposition on PEMs significantly diminishes after annealing. Cell adhesion, measured by the projected average cell spreading and focal contact formation, is remarkably improved for the annealed PEMs.